---- MANOMETRIC TECHNIQUES a manual describing methods applicable to the study of tissue metabolism FOURTH EDITION, 1964 » y ref 5. Benton Le W. W. UMBREIT RH. BURRIS J. F. STAUFFER Chapters of Specialized Techniques by ¥ M, J. JOHNSON, V. R. POTTER ai ‘AND W. C. SCHNEIDER 2 ed v Contibutions by [—~ J. A. BAIN, H. BEINERT, P. P, COHEN, HF, DeLUCA, H. A. LARDY, G. A, LePAGE I AND G. A. PALMER | Su ah Sl + Ming etChapter | THE WARBURG CONSTANT VOLUME RESPIROMETER INTRODUCTION Manometric methods for estimating exchange of gases have been used tn the study of both chemical and bivlugical reactious for generations. A wide variety uf (eclulgues have boon omployed and many typos of apparatus have boon devoloped. ‘The type of xcapire meter which has met with widest use is commonly called the "Warhurg" instrument, although, as pointed out by Warburg (1926), it was used before his adoption and modifica~ tion of it. In essence the present instrument is derived from a “'blood-gas manometer" described by Barcroft and Haldane (1902) or from that described by Brodie (1910). The resplrometer 1s based on the principle that at constant temperature and constant gas volume any changes in the amount of a gas can be measured by changes ist ils pressure. As the apparatus is most commonly applied to measurements of oxygen uptake, we shall first describe its principles in terms of oxygen uptake and later consider other mses of the instrament. APPARATUS flask sidearm sidearm stopper with gas vent venules well (fur alkali) M~ manometer proper R = fluid reservoir; adjustment of the screw clamp alters the evel of the fluid in the manometer T = three-way stopcock acum ‘The scale of the manometer ts graduated in centimeters (qum- bered) and in millimeters. Normally readings are recorded in millimeters to the nearest mm. or 0.5 mm. ‘The apparatus (fig. 1) consists of a detachable flask (F) some~ times equipped with one or more sidearms (8), attached toa manometer (M) containing a liquid of known density. The flask is immersed :n a water bath at a constant temperature, and betwoen readings tho system is shakon to promote a rapid gac exchange between the fluid and the gas phase. It is assumed Figure 1 that the temperature of the manometer, which is not immersed, ‘The Warburg constant does not differ greatly from that of the flask. Details of the volume respirometer apparatus have been described by Burk and Milner (1932), Dixon (1981), Perkins (1943), Warburg (1923, 1924, 1926) and others. Further details of shaking apparatus will be found in Chapter 8. ‘The manometer shown in figure 1 has an open and a closed end. A reference point on the closed side of the manometer (usually 150 or 250 mm.) is chosen, and the liquid in the closed arm of the manometer is always adjusted to this point before recording pressure changes.THE WARBURG CONSTANT VOLUME RESPIKUMETER GENERAL PRINCIPLES ‘10 measure an oxygen consuming reaction occurring in the flask, one turns the screw clamp on the fluid reservoir to adjust the manometer fluid to 2a0 mm. on the stopcock side of the manometer while the stopcock remains open, The slopcuck then is closed, and the reading of the open arm of the manometer ie recorded (accume that this is 249 mm. as in figure 2). After 10 minutes, the liquid has gone up in the closed arm and down in the open arm of the manometer as illustrated in the figure. One again adjusts the 29 pn Pe 2 wri osiTIon Posttion Bite to tinutes Figure 2 Diagram illustrating the determinatian of pressure change closed arm to 250 mm, and thus holds the volume of gas in the flask constant. ‘The reading on the open arm now 1s 220 mm. For both the initial and 10 minute readings the fluid in the closed arm of tho mano- meter was adjusted to 250 mm., but during the interval the reading on the open arm decreased from 249 to 220 mm, (29 mm.) as a result of oxygen consumption in the flask. If one knows the gas volume of the flask (Vg), the volume of fluid in the flask (Vg), the temperature of operation, the gaa being exchanged, and the density of the fluid in the manometer, it is possible to calculate the amount of gas used up (or gtven off), providing only one gas is being changed, (There are suethuds (u be de~ scribed later for handling alterations in the amount of more than one gas.) ‘The essence of the method is to hold the eas and fluid volumes constant and to measure tne decrease or increase in pressure when one gas alters in amount. DERIVATION AND MEANING OF FLASK CONSTANT fundamentally this consists of so calibrating the system that from the observed pressure changes one ean calculate the amount (in mm.” or micro liters (jl) at 0°C. and 760 mm. Pressure) of gao utilized or given off, The actual imelhud for calibrating the apparatus will be deserihed in Chapter 4 The following symbols are employed: etn the observed change in the manometer (open side) reading in mm, x= al. gas (O°C., 760 mm, Hg pressure), Ve - Volume of gas phace in flask including connecting tubes down ty Ue! reference point (150 ar 250 mm, on closed arm of manometer). vt P ‘Volume of fluid in vessel. Initial pressure in vessel of the gas involved in the determination. This is actually the partial pressure of the particular gas ina gas mixture, If this gas mixture contains water vapor, the partial pres- sure of gas involved in the determination will be tess than its partial pressure in tho dry condition. Hence if P is defined as dry fas, P - R should be used in equations involving moist gases. Po = standard pressure, which is 760 mm, Hg or 10,000 mm. Krebs! or Brodie's fluid, T = Temperature of bath in absolute degrees (= 273+ temp. in °C.). i